KR20140074017A - Parmaceutical Composition for Preventing or Treating Cancer Containing ABT-737 and Cafestol - Google Patents

Abstract

The present invention relates to a pharmaceutical composition containing ABT-737 and cafestol as active ingredients to prevent or treat cancer. The ABT-737 and cafestol have an excellent anticancer effect against Mcl-1 overexpressed cancer cells by inducing cancer cell-specific apoptosis if being used together, thereby being effectively used for preventing or treating cancer, specifically, kidney cancer.

Description

TECHNICAL FIELD The present invention relates to a pharmaceutical composition for preventing or treating cancer comprising ABT-737 and cafe stol as an active ingredient.

The present invention relates to a pharmaceutical composition for preventing or treating cancer comprising ABT-737 and cafestol as an active ingredient.

Malignant tumors that threaten human health, or cancer, are the leading cause of death worldwide, and are the most common cause of death in Western society after cardiovascular disease. In modern society, aging of population, increase of smoking population, and lung cancer due to air pollution are increasing, and as a result of westernization of eating habits, increase of environmental pollutants and increase of drinking water, colon cancer, breast cancer, prostate cancer Is on a steady increase trend. Therefore, it is urgently required to create an anticancer substance which can contribute to the promotion of human health, the improvement of a healthy life quality and the improvement of human health by enabling early prevention and treatment of cancer.

Tumors have a variety of mechanisms to inhibit intracellular apoptosis. Among them, Bcl-2 class, known as anti-apoptotic protein, is the most well-known. In fact, Bcl-2 class proteins Bcl-2, Bcl-x _L And Mcl-1 are known to be highly expressed.

Accordingly, many drugs that inhibit the expression of such Bcl-2 class proteins have been developed, and in fact, therapies for inducing the death of tumor cells using such drugs have been found. Among them, ABT-737 (CAS registry number: 852808-04-9, C ₄₂ H ₄₅ ClN ₆ O ₅ S ₂ ), which is known to inhibit the activation of Bcl-2 class proteins, has been attracted attention by inducing the death of tumor cells effectively Nature 435, 677-681 (2 June 2005)), it was found that ABT-737 alone can not induce apoptosis when Mcl-1 is overexpressed because the affinity to Mcl-1 is relatively low. Thus, ABT-737 alone has some effects on some small-cell lung cancer cells and leukemia cells, but it has no effect on other cancer cells including kidney cancer cell lines.

Therefore, studies have been conducted to develop a new effective cancer treatment agent that targets a cell pathway by using a substance such as ARC, which is an anti-cancer agent that targets Mcl-1, in combination with ABT-737 (UG Bhat, et al ., ARC Synergizes with ABT-737 to Induce Apoptosis in Human Cancer Cells, Mol Cancer Ther. June 2010 9: 1688-1696).

Accordingly, the present invention provides a novel anticancer agent which can be applied to cancer cells overexpressing Mcl-1.

In order to solve the above problems, the present invention provides a pharmaceutical composition for preventing or treating cancer comprising ABT-737 and cafestol as an active ingredient.

ABT-737 and cafestol exhibit anticancer activity through induction of apoptosis of cancer cells overexpressing Mcl-1, and the cancer overexpressing Mcl-1 may be kidney cancer.

When ABT-737 and cafestol were used in combination, ABT-737 and cafestol induced cancer cell-specific apoptosis and showed excellent anticancer activity in Mcl-1 overexpressed cancer cells. Especially for the prevention and treatment of kidney cancer.

Figure 1 is a Western blot result showing that expression of Mcl-1 in Bcl-2 class proteins in renal cancer cells is specifically reduced by caffeol treatment.
FIG. 2 shows experimental results of ABT-737 and caffeostol on cancer cell-specific killing induction. A is a photograph of cell morphological change, B is a flow cytometry analysis result, and C is a Western blot result of the cell death marker PLC-γ1.
FIG. 3 shows the results of an experiment in which the antitumor activity of ABT-737 and caffeostol was examined in vivo after cancer cell xenotransplantation. A is the mouse and isolated tumor photographs of the tumor size, and B is the photograph of the TUNEL assay results.

The present invention provides a pharmaceutical composition for preventing or treating cancer comprising ABT-737 and cafestol as an active ingredient.

The inventors of the present invention have studied ABT-737 for application to cancer cells overexpressing Mcl-1, and when used together with cafestol, induce the death of cancer cells overexpressing Mcl-1 effectively, The present inventors have completed the present invention by confirming that the anticancer activity is much superior to the case where each compound is treated alone.

In one embodiment of the present invention, ABT-737 may be a compound represented by the following formula (1).

[Chemical Formula 1]

In another embodiment of the present invention, cafestol may be a compound represented by the following formula (2) as a coffee oil ingredient contained in coffee.

(2)

The ABT-737 and cafestol compounds may be used in the form of a pharmaceutically acceptable salt. As the salt, acid addition salt formed by a pharmaceutically acceptable free acid is useful. As the free acid, inorganic acid and organic acid can be used. As the inorganic acid, hydrochloric acid, bromic acid, sulfuric acid, sulfurous acid, phosphoric acid and the like can be used. As the organic acid, citric acid, acetic acid, maleic acid, fumaric acid, , Acetic acid, glycolic acid, succinic acid, tartaric acid, 4-toluenesulfonic acid, galacturonic acid, embonic acid, glutamic acid, citric acid and arpartic acid. Preferably, hydrochloric acid is used as the inorganic acid, and methanesulfonic acid is used as the organic acid.

As can be seen from the following examples, the ABT-737 and cafestol compounds according to the present invention inhibit the proliferation of kidney cancer cells at a low micromolar concentration and induce a cell death-inducing mechanism that inhibits Mcl-1 And exhibits excellent anticancer activity. Thus, it can be used as an active ingredient of an anticancer agent. Accordingly, the present invention provides a pharmaceutical composition for preventing or treating cancer comprising ABT-737 and cafestol as an active ingredient, the use of ABT-737 and cafestol for the preparation of an anticancer agent, 737 and cafestol to a subject in need thereof.

In one embodiment of the present invention, the pharmaceutical composition for prevention or treatment of cancer comprising ABT-737 and cafestol as an active ingredient is prepared by mixing ABT-737 and cafestol with 100 parts by weight of the pharmaceutical composition, May be in the range of 0.01 to 90 parts by weight, 0.1 to 90 parts by weight, 1 to 90 parts by weight, or 10 to 90 parts by weight, but it is not limited thereto and may vary depending on the condition of the patient, .

In another embodiment of the present invention, the weight ratio of ABT-737 and cefstol in the pharmaceutical composition may be 1: (0.7-1.3), but is not limited thereto.

In another embodiment of the present invention, the pharmaceutical composition for preventing or treating cancer comprising ABT-737 and cafestol as an active ingredient may be a carrier, an excipient, a disintegrant, a sweetener, a coating agent, a swelling agent, And may further comprise one or more adjuvants selected from the group consisting of flavoring agents, flavoring agents, antioxidants, buffers, bacteriostats, diluents, dispersants, surfactants, binders and lubricants.

Specific examples of carriers, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, Cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. Solid formulations for oral administration may be in the form of tablets, pills, powders, granules, capsules These solid preparations can be prepared by mixing at least one excipient such as starch, calcium carbonate, sucrose or lactose, gelatin or the like in the composition. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions, syrups and the like, and various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included in addition to water and liquid paraffin which are commonly used simple diluents. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, suppositories, and the like. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. As the suppository base, witepsol, macrogol, tween 61, cacao paper, laurin, glycerogelatin and the like can be used.

In another embodiment of the present invention, the pharmaceutical composition for preventing or treating cancer comprising ABT-737 and cafestol as an active ingredient may be in the form of granules, powders, coated tablets, tablets, pills, capsules, Suppositories, gels, syrups, juices, suspensions, emulsions, drops, or liquids.

According to one embodiment of the present invention, the pharmaceutical composition may be administered orally, intraarterally, intraperitoneally, intramuscularly, intraarterally, intraperitoneally, intrasternally, transdermally, nasally, inhaled, topically, rectally, ≪ / RTI > can be administered to the subject in a conventional manner.

The preferred dose of the pharmaceutical composition for preventing or treating cancer comprising ABT-737 and cafestol as an active ingredient may be appropriately selected depending on the condition and body weight of the patient, the type and degree of the disease, the drug form, And may be suitably selected by those skilled in the art. According to one embodiment of the present invention, the daily dose may be 0.01 to 1,000 mg / kg, specifically 0.1 to 1,000 mg / kg, more specifically 0.1 to 100 mg / kg, though it is not limited thereto. The administration may be performed once a day or divided into several times, and thus the scope of the present invention is not limited thereto.

In the present invention, the 'subject' may be a mammal including a human, but is not limited thereto.

In one embodiment of the invention, the cancer may be a solid cancer, and more specifically ABT-737 and cafestol according to the present invention may be used for the treatment of brain tumors, low-grade astrocytoma, We report a case of high-grade astrocytoma, pituitary adenoma, meningioma, CNS lymphoma, oligodendroglioma, craniopharyngioma, ependymoma, brain tumor (Nasopharyngeal tumor), salivary gland (nasopharyngeal carcinoma), pancreatic cancer, pancreatic cancer, ovarian cancer, ovarian cancer, head and neck tumor, head and neck tumor, laryngeal cancer, tumor, small cell lung cancer, non-small cell lung cancer, hypopharyngeal cancer, thyroid cancer, oral cavity tumor, Chest Tumor, , Thymoma (Thymoma), mediastinal tumors (Med breast cancer, Abdomen-pelvis tumor, Stomach cancer, Hepatoma, Gall bladder cancer, Esophageal cancer, Esophageal cancer, Breast cancer, Male breast cancer, Biliary tract cancer, pancreatic cancer, small intestinal tumor, large intestinal tumor, anal cancer, bladder cancer, kidney cancer Renal cell carcinoma, male genital cancer, penile cancer, prostate cancer, female genital cancer, cervix cancer, endometrial cancer, For the treatment of endometrial cancer, ovarian cancer, uterine sarcoma, vaginal cancer, female vaginal cancer, female urethral cancer or skin cancer. It is preferable to use it. More preferably a renal cancer, a breast cancer, or a brain tumor, and still more preferably can be used for the treatment of kidney cancer, but is not limited thereto.

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail with reference to the following examples. However, the following examples are intended to illustrate the contents of the present invention, but the scope of the present invention is not limited to the following examples. Embodiments of the present invention are provided to more fully describe the present invention to those skilled in the art.

< Example  1> Cafe stall  Due to processing Mcl -1 decreased expression

Kidney cancer cells were cultured in a 6-well plate (4 × 10 ⁵ cells / ml) in 2 ml of CO ₂ incubator at 37 ° C for 24 hours, and then Cafestol was treated at 10, 30 and 50 mM . Western blotting was performed to confirm the expression of Mcl-1 in Bcl-2 class proteins. Cells treated with caffeine stones were disrupted with cell lysis buffer (50 mM Tris-Cl, 1 mM EGTA, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, pH 7.5) After SDS-PAGE, the separated proteins were transferred to Immobilon-P membrane at 90 volts for 1 hour. Protein-transferred membrane was reacted with 5% skim milk for 30 minutes at room temperature. Each of the individual antibodies was treated and reacted overnight at room temperature. The cells were washed 6 times with TTBS every 5 minutes, then reacted with secondary antibody with horse radish peroxidase (HRP) for 1 hour, and again with TTBS for 6 times And washed. Enhanced chemiluminescence (ECL) was confirmed by X-ray film using Western blotting kit. The results are shown in Fig.

1, it was confirmed that the expression of Mcl-1 in the Bcl-2 class protein in renal cancer cells was specifically reduced by the caffeostol treatment.

< Example  2> ABT -737 and Cafe stall Review of Cancer Cell Specific Action

ABT-737 and caffeostole were treated with ABT-737 alone or in combination with kidney cancer cells and normal cells in order to determine whether apoptosis by ABT-737 or cafe stole was a cancer cell-specific phenomenon. Cell morphology, flow cytometry, Cell death was confirmed by Western blotting.

Kidney cancer cells and normal cells were cultured in a 6-well plate at 4 × 10 ⁵ cells / ml for 24 hours in a CO ₂ incubator at 37 ° C for 24 hours, and then treated with ABT-737 alone or in combination with caffeostol The morphological changes of the cells were confirmed by optical microscope. The results are shown in Fig. 2A. As shown in FIG. 2A, when ABT-737 and caffeostol were combined in kidney cancer cells, cell morphology was changed, and it was confirmed that cell death was induced by breakage of cell shape, and no morphological change was observed in normal cells.

After adding 100 ml of PBS to the cells, 200 ml of 100% ethanol was added thereto while vortexing and reacted at 4 ° C for 1 hour. After removing the supernatant by centrifugation, 250 ml of RNase was added and incubated at 37 ° C for 30 minutes. Then, the same amount of PI (Propidium Iodide) was added and the cells were killed with a FACScan flow cytometer sub G1). The results are shown in Fig. 2B. Referring to FIG. 2B, it was found that apoptosis was increased to about 25% when the two drugs were treated in combination with ABT-737 and apolipoprotein alone in the treatment of kidney cancer cells. On the other hand, in normal cells, cell death was not induced by the combination of two drugs.

Fig. 2C shows the result of confirming PLC-γ1 as a marker of cell death through Western blotting. Referring to FIG. 2C, it was confirmed that a cleaved form of PLC-γ1 was formed when ABT-737 and caffeostol were specifically incorporated into kidney cancer cells.

These results indicate that cell death by the combination of ABT-737 and caffeostole is a cancer cell-specific phenomenon.

< Example  3> ABT -737 and Cafe stall in vivo  Review of anti-cancer activity

The mice were xenografted with 1 × 10 ⁶ cells / 100 ml of kidney cancer cells overexpressing Mcl-1. After 2 weeks, 75 mg / kg ABT-737 and 75 mg / kg caffeostole were administered twice a week Were injected into the abdominal cavity alone or in combination. The size of tumor through differentiation and death control is shown in Fig. Referring to FIG. 3A, the effect of reducing ABT-737 or caffeostol alone was small, but the tumor size was significantly decreased when ABT-737 and CAPESOL were combined.

The isolated tumor was rapidly cooled with OCT compound and cut to a thickness of 20 mm using cryostat, which is a low-temperature maintenance device. The tumor was stained with TUNEL assay, which is one of the methods for confirming tumor death, The death of the tumor was confirmed. Green color is a phenomenon that appears when the tumor is killed. TUNEL positive cells appear and blue color is stained with Hoecst33342. The results are shown in Fig. 3B.

Referring to FIG. 3B, green color signals are hardly observed in ABT-737 or caffeostol alone group, but the signal of green color, TUNEL positive, is increased when ABT-737 and caffeostol are combined As can be seen, it was confirmed that the tumor death was increased.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that such detail is solved by the person skilled in the art without departing from the scope of the invention. will be. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Claims (7)

A pharmaceutical composition for preventing or treating cancer comprising ABT-737 and cafestol as an active ingredient. ABT-737 and cafestol exhibit anticancer activity through the induction of apoptosis of cancer cells overexpressing Mcl-1. The method according to claim 1,
The pharmaceutical composition for the prevention or treatment of cancer may be a carrier, an excipient, a disintegrant, a sweetener, a coating agent, a swelling agent, a lubricant, a lubricant, a flavoring agent, an antioxidant, a buffer, a bacteriostatic agent, a diluent, a dispersant, a surfactant, Wherein the composition further comprises at least one adjuvant selected from the group consisting of: The method according to claim 1,
The pharmaceutical composition for preventing or treating cancer may be selected from the group consisting of granules, powders, coated tablets, tablets, pills, capsules, suppositories, gels, syrups, juices, suspensions, emulsions, Wherein the pharmaceutical composition is for preventing or treating cancer. The method according to claim 1,
Wherein said cancer is a solid cancer. 6. The method of claim 5,
The solid tumor may be selected from the group consisting of brain tumor, benign astrocytoma, malignant astrocytoma, pituitary adenoma, meningioma, brain lymphoma, oligodendroglioma, intracranial lesion, ependymoma, brain tumor, head and neck tumor, laryngeal cancer, Cancer, breast cancer, breast cancer, gastric cancer, liver cancer, gallbladder cancer, biliary cancer, pancreatic cancer, small bowel cancer, pancreatic cancer, breast cancer, pancreatic cancer, cancer, hypopharyngeal cancer, thyroid cancer, oral cancer, thoracic tumor, small cell lung cancer, non- Ovarian cancer, uterine sarcoma, vaginal cancer, female germ cell cancer, female urethral cancer, colon cancer, colon cancer, rectal cancer, anal cancer, bladder cancer, kidney cancer, male genital tumor, penile cancer, prostate cancer, female genital tumor, cervical cancer, Or skin cancer. &Lt; / RTI &gt; 6. The method of claim 5,
Wherein the solid cancer is kidney cancer.

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